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Abstract
Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Primarily on the basis of automation criteria, we have developed the BUME method, a novel chloroform-free total lipid extraction method for blood plasma compatible with standard 96-well robots. In only 60 min, 96 samples can be automatically extracted with lipid profiles of commonly analyzed lipid classes almost identically and with absolute recoveries similar or better to what is obtained using the chloroform-based reference method. Lipid recoveries were linear from 10-100 µl plasma for all investigated lipids using the developed extraction protocol. The BUME protocol includes an initial one-phase extraction of plasma into 300 µl butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into 300 µl heptane:ethyl acetate (3:1) using 300 µl 1% acetic acid as buffer. The lipids investigated included the most abundant plasma lipid classes (e.g., cholesterol ester, free cholesterol, triacylglycerol, phosphatidylcholine, and sphingomyelin) as well as less abundant but biologically important lipid classes, including ceramide, diacylglycerol, and lyso-phospholipids. This novel method has been successfully implemented in our laboratory and is now used daily. We conclude that the fully automated, high-throughput BUME method can replace chloroform-based methods, saving both human and environmental resources.
Highlights
Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research
It is important that the lipid extract that is injected on the HPLC or infused into the mass spectrometer is pure; it is important that interfering substances and particles are removed
BUME mixtures as solvent 1 worked very well in combination with solvent 2 and the buffer and fulfilled all requirements postulated in Table 1 for automation
Summary
Lipid extraction from biological samples is a critical and often tedious preanalytical step in lipid research. Two of the most commonly used methods for extracting and purifying lipids are the Folch [4] and Bligh and Dyer [5] procedures Even though both of these methods are highly efficient for extracting lipids within a wide range of hydrophobicity, they are associated with important disadvantages. Both of these methods are based on chloroform, which in a mixture with methanol, is the primary solvent for solubilization. A method based on methyl-tert-butyl ether (MTBE) was published by Matyash et al [8] Even though this method was shown to have a lipid-containing upper phase and to be well-suited for total lipid extraction, the method has a high solvent-to-sample ratio, making automation in the 96-well format challenging
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